In Vitro Depletion of CD4⁺CD25⁺ T Regulatory (T_reg) Cells with Denileukin Diftitox (DAB₃₈₉IL-2) Increases T Cell Alloreactivity.

We hypothesize that immune tolerance after allogeneic hematopoietic cell transplantation is maintained by an active cellular mechanism that includes T_reg CD4⁺CD25⁺ cells. In the dog model of mixed hematopoietic chimerism, adoptive immunotherapy with donor lymphocyte infusion (DLI) that successfully converted recipients to all-donor chimerism required the breaking of immune tolerance to target antigens. We reasoned that depletion of T_reg cells might break tolerance and could improve the therapeutic efficacy of subsequent DLI. Denileukin diftitox (DAB₃₈₉IL-2, Ontak) is a fusion protein consisting of the translocation and ADP-ribosylase domains of diphtheria toxin and interleukin 2. DAB₃₈₉IL-2 binds to the high affinity IL-2 receptor (CD25, CD122 and CD132) expressed on the cell surface of activated T cells. Some T_reg cells express the high affinity IL-2 receptor. Although DAB₃₈₉IL-2 has been shown to deplete alloreactive T cells, we asked if DAB₃₈₉IL-2 could deplete T_reg cells without depleting alloreactive T cells in dog mixed lymphocyte cultures (MLC). First we asked if DAB₃₈₉IL-2 could induce specific cell death of activated T cells. Five days after initiation of a one-way, dog leukocyte antigen (DLA)-mismatched MLC, DAB₃₈₉IL-2 was added to the MLC for 24 hours and cells were pulsed with ³H-thymidine. At 10⁻¹¹ and 10⁻¹⁰ M [DAB₃₈₉IL-2] there was 34% and 92% inhibition of cell proliferation, respectively. To determine if DAB₃₈₉IL-2 could deplete T_reg cells, we established DLA-mismatched MLC (n=5) with cyclosporine (CSP) 400ng/mL (Martin, PJ et al. BBMT, 2004) and added DAB₃₈₉IL-2 in half-log increments from 10⁻¹¹ to 10^−9.5 M on day 4 of MLC. On day 5, culture medium was changed to remove DAB₃₈₉IL-2 and cells were analyzed for expression of CD4 and CD25. Flow cytometric analysis showed that CSP treated MLC had reduced CD4⁺CD25^bright and CD4⁺CD25^dim by 90 ± 3.3% and 64 ± 10%, respectively, compared to no CSP. DAB₃₈₉IL-2 [10⁻¹¹ M] treatment of MLC with CSP decreased CD4⁺CD25^bright and ^dim populations 31 ± 12% and 30 ± 4%, respectively, compared to no DAB₃₈₉IL-2. On day 10 of the culture, cells were tested in secondary (2°) MLC to determine the effect of depleting CD4⁺CD25⁺ cells with DAB₃₈₉IL-2. 2° MLC was performed without the addition CSP or DAB₃₈₉IL-2. After 3 days of 2° MLC, cells were assayed for proliferation. Compared to controls with no CSP or DAB₃₈₉IL-2 treatment during 1° MLC, depletion of CD4⁺CD25⁺ cells with DAB₃₈₉IL-2 increased 2° allo-specific proliferation 36.2 ± 26%. When CSP was present in the 1° MLC, DAB₃₈₉IL-2 treatment on day 4 increased subsequent 2° MLC allo-specific T cell proliferation 86 ± 22%. These results are consistent with the hypothesis that DAB₃₈₉IL-2 can efficiently eliminate T_reg (CD4⁺CD25⁺) cells, particularly when T_reg cells are generated in the presence of CSP.